Article Figures & Data
Figures
- Figure 1.
Expression of bovine 7OR-PrP and 6OR-PrP proteins in heterozygous (mu +/-bo +/-) boTg lines. Immunoblotting brain extracts from bo7ORTg mouse lines 001, 006, 007, 012, and 027 and bo6ORTg mouse lines 113 and 135 using mAb 2A11 (A) or FH11 (B). Serial dilutions of homozygous (mu-/-bo+/+) bo6ORTg (113, 135, and 110 mouse lines), bo7ORTg (007 and 006 mouse lines), and bovine brain homogenates were analyzed by Western blotting using mAb 2A11 (C). Bo, Cow brain extract; Mo, B6CBAx129/Ola brain extract. Equivalent amounts of total protein were loaded on each lane. Relative molecular mass is expressed in kilodaltons.
- Figure 2.
Onset of clinical signs after inoculating BSE in different bovine transgenic mice. In the columns (A) or Kaplan-Meier curves (B), incubation times are compared among bo6ORTg and bo7ORTg lines (showing threefold or sixfold the PrPC levels detected in bovine brain homogenates) inoculated with BSE1, BSE2, and first passage BSE1 in bo7ORTg (bo7ORTgBSE1). Values are expressed as days after inoculation ± SD.
- Figure 3.
Survival times after BSE inoculation in different bovine transgenic mice. In the columns (A) or Kaplan-Meier curves (B), survival times are compared among bo6ORTg and bo7ORTg (showing threefold or sixfold the PrPC levels detected in bovine brain homogenates) inoculated with BSE1, BSE2, and first passage BSE1 in bo7ORTg (bo7ORBSE1). Values are expressed as days after inoculation ± SD.
- Figure 4.
A, B, Histopathological changes consisting in vacuolation in the deeper layers of the cerebral cortex and the hippocampus (1) and the cerebellar white matter (4); GFAP immundetection in the deeper layers of the cerebral cortex and the hippocampus (2) and cerebellar white matter (5) showing astrogliosis and astrocytosis; and PrPres immunodetection using mAb 2A11 in the deeper layers of the cerebral cortex and the hippocampus (3) and the cerebellar white matter (6) of bo6ORTg (A) or bo7ORTg (B) mice inoculated with BSE2 inoculum (∼200 d after inoculation). C, D, Histopathological changes consisting in vacuolation in the deeper layers of the cerebral cortex and the hippocampus (1) and the cerebellar white matter (4); GFAP immundetection in the deeper layers of the cerebral cortex and the hippocampus (2) and cerebellar white matter (5) showing astrogliosis and astrocytosis; and PrPres immunodetection using mAb 2A11 in the deeper layers of the cerebral cortex and the hippocampus (3) and the cerebellar white matter (6) of boORTg mice inoculated with the BSE1 inoculum passage one in bo6ORTg (C) or bo7ORTg mice inoculated with BSE1 inoculum passage once in bo7ORTg mice (D) (∼250 d after inoculation). E, F, Histopathology (1 and 3) and PrPres immunodetection (2 and 4) of bo6ORTg noninoculated mice as negative controls, showing no changes (E). Histopthological changes consisting in vacuolation (1) and PrPres immunodetection (2) in the brainstem of BSE-infected cattle as positive control (F). Scale bar, 50 μm.
- Figure 5.
A, Comparative studies using brain homogenates from silent carriers as inoculum. Bo6ORTg and bo7ORTg mice were inoculated with bo6ORTgBSE1120d (120) and bo6ORTgBSE1150d (150) (two pools of brain homogenates from bo6ORTg mice killed at 120 or 150 d after inoculation with BSE1 inoculum). Both inocula contained amounts of PrPres undetectable by conventional immunoblot analysis. Tg mice were killed at 330 d after inoculation, and PrPres from brain samples were analyzed by Western blot after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrPres at the indicated time after inoculation. B, Comparative kinetics of PrPres detection after BSE2 inoculation in bo6ORTg and bo7ORTg mice. Bovine transgenic mice expressing 6OR-PrP or 7OR-PrP were inoculated with BSE2 inoculum and killed at the indicated time points, irrespective of the onset of neurological signs. PrPres in brain samples were analyzed by Western blotting after proteinase K (20 μg/ml) treatment. Columns indicate the percentage of Tg mice showing PrPres at the indicated time after inoculation.
Tables
Transgene expressiona Incubation time (days ± SEM) Recipient Inoculum Illness Death Numberb Non-Tg (B6xCBAx1290la) BSE1 1x 614 = 26 656 ± 30 12 Non-Tg (prnp−/−) BSE1 0x 599 = 14 688 ± 35 8 bo6ORTg110+/+ BSE1 8x 272 ± 8 326 ± 18 13 bo6ORTg110+/− BSE1 4x 311 = 17 359 ± 15 11 bo6ORTg113+/+ BSE1 3x 352 = 10 410 ± 7 10 bo6ORTg135+/+ BSE1 6x 290 = 10 381 ± 18 6 bo7ORTg006+/+ BSE1 6x 216 ± 8 279 ± 14 9 bo7ORTg006+/− BSE1 3x 257 ± 7 313 ± 7 4 bo7ORTg007+/+ BSE1 4x 237 ± 4 286 ± 5 4 bo7ORTg007+/− BSE1 2x 325 = 10 376 ± 14 6 bo6ORTg110+/+ BSE2 8x 251 = 20 308 ± 5 5 bo6ORTg113+/+ BSE2 3x 328 ± 5 377 ± 8 7 bo6ORTg135+/+ BSE2 6x 257 = 11 324 ± 25 7 bo7ORTg006+/+ BSE2 6x 156 ± 5 175 ± 10 11 bo7ORTg006+/− BSE2 3x 235 ± 3 253 ± 14 5 bo7ORTg007+/+ BSE2 4x 218 ± 7 237 ± 7 4 bo7ORTg007+/− BSE2 2x 294 ± 5 337 ± 26 4 bo6ORTg110+/+ bo7ORTgBSE1 8x 205 ± 4 291 ± 18 6 bo6ORTg113+/+ bo7ORTgBSE1 3x 280 ± 6 319 ± 9 9 bo6ORTg135+/+ bo7ORTgBSE1 6x 259 = 12 323 ± 1 4 bo7ORTg006+/+ bo7ORTgBSE1 6x 142 ± 3 171 ± 5 8 bo7ORTg007+/+ bo7ORTgBSE1 4x 279 = 11 331 ± 20 8 bo6ORTg110+/+ PBS inoculated 8x 490 ± 5 599 ± 27 4 bo6ORTg113+/+ PBS inoculated 3x 491 ± 5 581 ± 39 4 bo6ORTg135+/+ PBS inoculated 6x 461 = 10 607 ± 49 4 bo7ORTg006+/+ PBS inoculated 6x 561 ± 36 589 ± 34 6 bo7ORTg007+/+ PBS inoculated 4x >550 >550 6
Supplementary material
Files in this Data Supplement:
- Supplementary Fig. 1 - Supplementary Figure 1. Diagram showing the 7OR-PrP gene construct. The 6OR-PrP gene, previously cut with the NcoI restriction enzyme to eliminate three OR, was used to insert 4 artificial octarepeats in tandem. These OR were generated by specific oligonucleotides with extremes compatible with NcoI (see Materials and Methods).
- supplementary Fig. 2 - Supplementary Figure 2. Quantification of the PrPres contents of different inocula. Serial dilutions of brain homogenates were analysed by Western blotting using MAb 2A11. BSE1: pool of BSE material (TSE/08/59) derived from the brainstems of 49 BSE infected cattle and supplied by the VLA; BSE2: inoculum derived from one BSE infected brainstem supplied by the VLA; bo7ORTgBSE1: pools from a first passage of BSE1 inoculum in bo7ORTg mice. Monoclonal antibody 2A11 was used at a 1:1500 dilution. Relative molecular mass in kilodaltons.
- supplementary Fig. 3 - Supplementary Figure 3. A: Brain homogenates from six bo6ORTg or bo7ORTg mice were solubilized in extraction buffer. Samples were pre-cleared by centrifugation and the supernatant was ultracentrifuged after treatment with 0, 1 and 3 M GdN HCl. Soluble (S) and insoluble (I) proteins were subsequently analysed by SDS-Page and Western blotting. B: Western blot analysis of brain homogenates from bo6ORTg or bo7ORTg mice inoculated with: BSE1, BSE2 or first passage BSE1 in bo7ORTg (bo7ORTgBSE1) before (p0) or after passage in bo6ORTg or bo7ORTg mice (p1 or p2). Samples were previously digested with proteinase-K (20 �g/ml). Monoclonal antibody 2A11 was used at a 1:1500 dilution. Molecular weight in kilodaltons.
